Adaptation Mechanism Of Ectomycorrhizal Pinus Massoniana Seedlings To Low Phosphorus Stress

Phosphorus（P）is an essential mineral required for plant growth and development.It is involved in photosynthesis,energy conversion,and signal transduction and plays an important role in plant metabolism.P deficiency will inhibit the growth of plants,reduce the yield of agriculture and forestry,and affect the healthy development of agriculture and forestry.P deficiency has gradually become the primary factor limiting forest productivity.At present,P fertilizers are mainly used to promote the growth of plants,but a large amount usage of P fertilizer accelerates the depletion of P resources,and causes environmental pollution.Therefore,exploring the advantages of plants to improve the uptake and utilization of P is particularly important.Pinus massoniana is a tree species that is native to southern China and primarily grows in subtropical and tropical regions,and is a typical ectomycorrhizal（ECM）tree species.P.massoniana has high utilization value and has become one of the most important tree species for afforestation in south China.It is often used as the main pioneer tree for vegetation restoration in barren mountains and wastelands,and plays an important role in forestry production.However,the growth of P.massoniana is often affected by P deficiency,resulting in the reduction of stand quality and the formation of low-efficiency forest.Studies have shown that inoculation of ECM fungi（ECMF）can promote plant growth and enhance stress resistance.Therefore,Suillus bovinus（Sb）,S.luteus（Sl）and Scleroderma citrinum（Sc）were used as materials to study their response to P and its promotion effect on growth.Then,the growth,physiological response,nutrient absorption and accumulate,characteristics of root exudates in inoculated and uninoculated P.massoniana seedlings were studied by sand culture pot experiment under different P supply levels.The transcription level of P.massoniana seedlings under low P stress also were studied.At the same time,growth physiology and activation effect of soil P under different insoluble P supply levels in P.massoniana seedlings inoculated Sc and uninoculated seedlings were studied.All the studies were to explore the response mechanism of P.massoniana to P stress and the mechanism of ECMF improving plant to low P tolerance.The main results obtained were as follows:1.Response of ECMF to P supply and the symbiotic effect on P.massoniana seedlingsP deficiency in medium had no significant effect on Sb growth,but significantly reduced the growth of Sl and Sc.The weekly increment of colony diameter increased in Sb,decreased in Sl,and increased firstly and then decreased in Sc with the culture time.The daily growth rate was Sc>Sl>Sb in the same P supply level,and the differences were significant.The utilization of Sc in different forms of insoluble phosphate was found that Sc grew best on the medium containing Al PO₄,Fe PO₄·2H₂O,and with a higher biomass,mycelium had higher N and P contents and medium had a lower p H value.Sc grew better on the medium containing KH₂PO₄.Sc grew little on the medium containing Ca₃（PO₄）₂,and the p H value increased 0.65.The Sb,Sl and Sc all could form symbiotic relationship with P.massoniana and promoted the growth of P.massoniana.The activities of POD,CAT,soluble protein were significantly increased in P.massoniana seedling inoculated with Sb and Sl.The activities of SOD and ACP,soluble sugar content were significantly increased in P.massoniana seedling inoculated with Sc.The K content in roots and the P content in leaf of P.massoniana seedling inoculated with ECMF were higher than that in the seedling without inoculated.The results showed that inoculation of ECMF promoted the growth of P.massoniana seedling by improving the antioxidant capacity and enhancing the absorption of nutrient elements,but the effects were different.In general,Sc and Sb are preferred.2.Responses of P.massoniana mycorrhizal seedling to P supply levelsThe growth,antioxidant system,osmotic regulation system,characteristics of root exudates,nutrient absorption and accumulation,and changes of transcription level of P.massoniana seedlings among inoculated with Sb,Sl and Sc and uninoculated seedlings under4 P supply levels were significantly different.（1）The net increment of height,ground diameter in mycorrhizal and non-mycorrhizal of P.massoniana seedling decreased with test time extension under 4 P supply levels.The total increment of height and ground diameter of P.massoniana seedling reduced with the decreased of P level,and with a lower biomass at low P supply level.The total biomass of mycorrhizal seedling under P1 and CK treatments were significantly higher than non-mycorrhizal seedling.The results showed that ECMF promoted the growth of P.massoniana seedlings under low P and normal P supply.The activities of ACP,SOD,POD and CAT,and MDA and proline contents of mycorrhizal and non-mycorrhizal of P.massoniana seedling under P1 treatment was higher than that under CK treatment,but the variation under P2 and P3 treatment are related to the types of seedlings.Moreover,the activities of ACP,SOD,POD and CAT,and the content of MDA and proline in mycorrhizal and non-mycorrhizal P.massoniana seedling under 4 P supply levels increased with time.The soluble sugar content in P1 treatment was significantly higher than that in CK treatment,and firstly decreased and then increased with the time extension under 4 P supply levels.The soluble protein content firstly increased and then decreased with the time under 4 P supply levels.Soluble protein content was significantly lower in P1 treatment than that in CK treatment.There was no significant difference in the soluble sugar and protein content of mycorrhizal and non-mycorrhizal of P.massoniana seedling among P2,P3 and CK treatments.These results indicated that low levels P supply caused oxidative stress on P.massoniana seedling,stimulated the response of antioxidant system,and affected sugar transport and protein synthesis.In P1 treatment,the activities of ACP,SOD and POD,and the contents of proline and soluble protein in the mycorrhizal seedling were higher than those in the non-mycorrhizal seedling,while the contents of MDA and soluble sugar were lower than those in the non-mycorrhizal P.massoniana seedling.However,the physiological responses of seedlings inoculated with Sb,Sl and Sc under P2 and P3 treatments were different.The results indicated that ECMF could improve the efficiency of P utilization,alleviate the oxidative damage caused by low P stress on P.massoniana seedling,and reduce the effect on sugar transport and protein synthesis.However,the alleviating effect was highest in P1treatment,the effects of Sb and Sc were better in generally.（2）The N,P and K contents and uptake of P.massoniana seedlings decreased in lower P supply in generally.This indicated that the low level of P supply reduced the nutrients absorption of P.massoniana.The Sb,Sl and Sc significantly increased the N content in root,P content in stem,P and K contents in leaf of P.massoniana seedlings in P1 treatment.The Sc significantly increased the P contents in root and the N content stem of P.massoniana seedlings in P1treatment.However,this stimulative effect is smaller under P2,P3,and CK treatments.The Sb,Sl and Sc significantly increased the absorb of N,P and K in root,stem,leaf of P.massoniana seedlings and increased the P utilization rate of leaves under P1 and CK treatments.It indicated that ECMF could improve the nutrient content and uptake of P.massoniana seedlings under low P stress and normal P supply,and Sc was generally better.（3）The root activity in uninoculated seedling and seedlings inoculated with Sl were significantly reduced under P1 treatment,but without significant changes in seedlings inoculated Sb and Sc.P2 and P3 treatments didn’t have significant effect on the root activity in uninoculated and inoculated seedling.The root length of uninoculated seedling,the roots surface area,volume,tips and forks of seedling inoculated with Sc were significant reduced under P1 treatment.There were no significant effects on the root growth of P.massoniana under P2 and P3 treatments.The Sb,Sl and Sc significantly increased the root activity of P.massoniana seedlings under P1 treatment and promoted the growth roots of P.massoniana seedlings under 4 P supply levels and Sb was better overall.ECMF and P supply level affected the composition and content of root secretion of P.massoniana seedlings,and had a significant effect on the secretion and content of organic acids,but the effect was different with the P supply level and the type of ECMF.The relative content of acids in the secretion of seedlings inoculated with Sb and Sc was higher than that in uninoculated seedlings under P1treatment.（4）P.massoniana seedlings could adapt to low P stress by accelerating signal transduction,improving P metabolism,regulating hormone signal transduction,reducing photosynthesis,and promoting the synthesis of secondary metabolites,but this regulation ability differed between mycorrhizal and non-mycorrhizal seedlings.Mycorrhizal seedlings enhanced the performance of P.massoniana under low P stress by up-regulation the expression of related genes in the photosynthesis pathways,phenylpropanoid biosynthesis pathways,and increased the number of transcription factors.The expression of differential genes in mycorrhizal seedlings increased with time under low P stress.Mycorrhizal seedlings also enhanced plant adaptation to low P stress by regulating the expression of genes related to ethylene signaling.3.The activation and utilization of insoluble phosphate in P.massoniana mycorrhizal and non-mycorrhizal seedlingsMycorrhizal seedlings could utilize insoluble phosphates in soil.Low levels of insoluble phosphate supply could stimulate the increase of O^2-,H₂O₂and MDA contents in P.massoniana seedlings,and reduce the contents of chlorophyll a,b,total chlorophyll and carotenoids,the total P and inorganic P contents in roots,stems and leaves,affected sugar transport and protein synthesis,and ultimately inhibited the growth of P.massoniana seedlings and reduced biomass accumulation.P.massoniana seedlings could respond to P deficiency by increasing antioxidant enzyme activity and antioxidant content.P.massoniana seedlings inoculated Sc had lower ROS content,higher P content and absorb content in roots,stems and leaves,higher photosynthetic pigment content and the growth of P.massoniana seedlings was less inhibited and the biomass was relatively higher.Therefore,Sc improving the adaptability of P.massoniana seedlings to low P stress by expanding the P absorption range,increasing the P absorption amount in the roots,stems and leaves,improving the photosynthetic capacity,reducing the damage of ROS.Therefore,ECMF improving the adaptability of P.massoniana seedlings to low P stress by expanding the P absorption range,increasing the activization of insoluble phosphates,improving the P utilization efficiency,the antioxidant ability,the photosynthetic ability and the sugar transport and protein synthesis capacity.